Passive immunotherapy with anti-amyloid beta peptide antibodies [anti-Abeta Abs] offers the benefits but not the toxicity of active immunization with Abeta peptide. The purpose of collaborative project is to develop and test proteolytic anti-Abeta Abs that not only bind Aa peptide but also cleave specifically the Aa peptide. Three approaches will be used to develop proteolytic anti-Abeta Abs: [1] selection of Ab fragments (single chain Fv fragments) from a phage library expressing the human Ab repertoire based on coordinated noncovalent recognition and irreversible binding of covalently reactive Abeta analogs (CRAs) containing chemical groups reactive with the nucleophiles in the active site of serine proteases. This approach has been validated for its capacity to identify Abs that combine specific antigen recognition with the ability to hydrolyze peptide bonds, [2] Improvement in anti-Abeta specificity by: (a) Randomizing the sequence of the heavy chain variable (VH) domain CDR3, a region governing Ab specificity, combined with covalent affinity selection using the CRAs; and (b) Pairing of light chain V domains expressing promiscuous proteolytic activity with VH domains derived from specific anti-Abeta Abs. In each case, the scFv clones will be tested for their capacity to neutralize Abeta peptide-mediated neurotoxicity in vitro. [3] Induction of proteolytic monoclonal anti-Abeta Abs by immunization with Abeta CRA, a strategy designed to permit clonal selection of B cells producing Abs with enhanced and specific anti-Abeta proteolytic activity will be studied. Determination of the Abeta cleavage site(s) will identify Ab clones capable of cleaving the determinant (residues 25-35) and oligomeric peptide states thought to be important in Abeta peptide toxicity using proteolytic and non-proteolytic Abs. The most active Abs will be tested in vivo for their capacity to inhibit cerebral amyloid deposits and cognitive decline in APP/PS1-transgenic mice. A RAG-1-deficient line of APP/PS1-transgenic mice will be used to test human proteolytic Abs, thus avoiding confounding murine immune responses to xenogeneic Abs. Proteolytic anti-Abeta Abs capable of preventing the deposition of Abeta and cognitive decline would be candidates for passive immunotherapy of humans with AD.